the immunoassay

The Immunoassay

Chad Chisolm, Jaclyn Holcombe, Matthew Shelnutt

Know that Toxicology is the branch of Forensic Science in which the immunoassay plays the greatest role.

Know that in the year 1959, the RIA, predecessor of modern immunoassays, was invented.

Know that the RIA was invented by Drs. Yalow and Berson.

Know that antibodies are proteins that are found in blood or other bodily fluids that are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

Know that an antigen is any substance that causes your immune system to produce antibodies against it. An antigen may be a foreign substance from the environment such as chemicals, bacteria, viruses, or pollen; they may also be produced inside the body.

Objectives

Objectives Cont’d

Know that a hybridoma is the hybrid of an antibody and a myleoma cell, which are cultured to produce antibodies.

Know that immunoassays are typically categorized as either competitive or noncompetitive.

Know that in noncompetitive immunoassays, the amount of labeled analyte is directly proportional to the amount of antigen; in the competitive, it is inversely proportional.

Know that heterogeneous immunoassays depend on the separation of unbound tracers before the bound signal is measured; homogeneous immunoassays do not require this separation because the signal is generated when the binding occurs.

Know that two specific types of immunoassays include the EMIT, RIA, ELISA, FPIA.

History and Background

• In the year 1959, Drs. Rosalyn Yalow & Soloman Berson invented the radioimmunoassay, which applied the use ofradioisotopes in themeasurement ofinsulin.

• The RIA is the predecessor of modern immunoassays.

Dr. Rosalyn Yalow became the firstfemale to win a Nobel Prize withher work on the radioimmunoassay.

History and Background Cont’d

• 1972 First Hepatitis RIA

• 1972 EMIT Assay

• 1975 Bead-based Assays

• 1978 Coated-tube assays

• 1985 HIV Immunoassay

• 1993 TroponinAssays

• 1997 Automated Cytokine Assays

(Evans)

Immunoassays at a Glance

• Immunoassays are a group of sensitive analytical tests that utilize very specific antibody/antigen complexes to produce a signal that can be measured and related to the concentration of a compound in solution (Wild). Immunoassays also produce qualitative data in terms of the presence (+) or absence (-) of a compound in the body.

• They are used in a lot of laboratories, including hospitals labs, and have been widely used in the special area of Forensic Toxicology to screen for drugs and other chemicals in the body.

Antibodies

• Antibodies have a generally common structure, but have regions that vary among them to accommodate the specific antigens (Abbott Diag.).

• Antibodies are produced by the B lymphocytes. They are glycoproteins belonging to the “immunoglobulin supergene family” (Wild) that are produced in response to a foreign substance in the body.

Antigens

• An antigen is a substance with the ability to induce an immunological response. They typically enter the body from an infection. They are recognized at their epitopes by B cells or by the T cell receptor on T cells (Lee).

• Proteins or glycoproteins make the best antigens because they are the best at stimulating antigen recognition molecules. Some immunoassays test for antigens, rather than antibodies (Lee).

Production of Antibodies

• The production of antibodies is an important process in the use of immunoassays because it is the antibody-antigen complexes that form that the device uses for its results. Antibodies can be called monoclonal or polyclonal, depending upon the technique used to produce it.

• Monoclonal antibodies involve these basic steps and result in very specific antibodies that bind only to one antigen epitope, which in turn reduces the occurrence of false positives in the immunoassay:

− A mouse (or rabbit) is immunized by being injected with an antigen; the antigen generates an antibody response in the animal.

− The mouse is sacrificed and the antibody forming cells are isolated from the mouse's spleen.

− Monoclonal antibodies are produced by fusing single antibody-forming cells with myeloma cells. The resulting cell is called a hybridoma. This hybridization makes the cells “immortal.”

− The hybridomas contain large amounts of antibodies, and can easily be cultured into populations of cells that contain identical antibodies. These antibodies are called "monoclonal antibodies" because they are produced by the identical offspring of a single, cloned antibody producing cell. (UCLA)

Production of Antibodies Cont’d

A diagram depicting the steps involved in monoclonal antibody production.

Production of Antibodies Cont’d

• Polyconal antibodies are more likely to produce a false positive because they are less specific to antigen epitopes and have varying binding affinities; they may bind of molecules similar to their antigens. Production includes these steps:

− A mouse (or rabbit) is immunized by being injected with an antigen; the antigen generates an antibody response in the animal.

− The animal is bled and the antibodies are collected; the blood contains a heterogenous mixture of antibodies of varying binding affinities and specificities. (Wild)

Labels in Immunoassays

• Immunoassays require the use of labeled materials in order to measure the amount of antigen or antibody present. A label is a molecule that will react as part of the assay, and in doing so produce a signal that can be measured in the solution. Examples of a label include a radioactive compound, or an enzyme that causes a change of color in a solution or its fluorescence (Wild).

Competitive Immunoassays

• The measurement of the analyte using the labels is broadly categorized into competitive and noncompetitive methods.

– In competitive formats, unlabelled analyte in the test sample is measured by its ability to compete with labeled antigen for a limited number of antibody binding sites (Bell). The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied. Thus, in a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is present. The amount of antigen in the test sample is inversely related to the amount of label measured in the competitive format (Abbott Diag.). As one increases, the other decreases.

Noncompetitive Immunoassays

– Noncompetitive (sandwhich) immunoassays generally provide the highest level of assay sensitivity and specificity. This format is referred to as a “sandwich” assay because the analyte is bound (sandwiched) between two highly specific antibody reagents.

– The reaction mixture typically includes an excess of labeled antibody, so that all drug/metabolite is bound. The amount of antibody-antigen complex is then measured to determine the amount of drug present in the sample. The measurement of labeled analyte, usually antibody, is directly proportional to the amount of antigen present in the sample.

Homogeneous VS Heterogeneous Methods

• Immunoassay methods that require separation of bound Ab-Ag* complex are referred to as heterogeneous immunoassays. Those that do not require separation are referred to as homogeneous immunoassays.

• Homogeneous methods have been generally applied to the measurement of small analytes such as abused and therapeutic drugs. Since homogeneous methods do not require the separation of the bound Ab-Ag* from the free Ag*, they are generally much easier and faster to perform.

Types of Immunoassays

• Radioimmunoassays (RIAs) utilize a radioactive label (usually 125I, 3H or 14C), which emits radiation that can be measured with a beta or gamma counter.

• Within the categories of competitive, noncompetitive, homogenous, and heterogeneous, there are specific types, which include:

Types of Immunoassays Cont’d

• In the Enzyme Multiplied Immunoassay (EMIT), the drug in the sample and the drug labeled with G6PD compete for antibody binding sites.

• Binding inhibits enzyme activity, while free enzyme remains active to interact with.

• Enzyme activity/absorbance is directly proportional to drug concentration.

Types of Immunoassays Cont’d

• Enzyme linked immunosorbant assay (ELISA): competitive, heterogeneous EIA

• Reaction components are absorbed or bound to the surface of a solid phase, commonly a well of a microtiter plate

• Absorbance is measured using a micro-plate reader

• Sample absorbance is inversely proportional to drug concentration

Types of Immunoassays Cont’d

• In the Fluorescent Polarized Immunoassay, the drug in the sample competes with fluorescein-labeled drug for antibody binding sites.

• Reaction mixture is excited by planepolarized light.

• As the tracer returns to a lower energy state, it emits light; polarization is measured.

• The polarization value of the sample is inversely proportional to analyte concentration.

Immunoassay Results

• Qualitative– Single point calibration at a specific cutoff– Results are either ‘positive’ or ‘negative’; (i.e.

above or below the cutoff)– Possible false positives; monoclonal antibodies

restrict this slightly.• Quantitative

– Provides numeric results that are an estimate of drug/compound concentration based on the measurement of labeled analyte in the solution, and taking into consideration the competitive/noncompetitive nature of the device.

– In terms of use on drugs, this is sometimes complicated by possible cross-reactivities.

Immunoassay Results Cont’d

Typical 4-parameter logistic graph for a competitive-format immunoassay.

Dose-response curve for a non-

competitive CL immunoassay.

A pregnancy test is an example of a commercially produced immunoassay that produces a positive or negative qualitative

response.

Standardization of Immunoassays

• The aim of standardization is to ensure that assays of the same analyte in the same samples, done at different places or at different times or both, can be readily compared (Masseyeff).

• International Standards are prepared by the National Institute for Biological Standards and Control in the UK (Wild).

• Preparation of standard proteins are purified, mixed with an inert carrier compound, divided and freeze-dried, and sent to laboratories (Wild).

Standardization of Immunassay Cont’d

• Known standards include:

– Cortisol

– Alphafetoprotein (AFP)

– Carcino-embryonic antigen (CEA)

– Human gonadotropin (hCG)

Cortisol(Wild)

Immunoassays and Forensic Science

• Forensic toxicology encompasses the determination of the presence and concentration of drugs, other xenobiotics and their metabolites in physiological fluids and organs and the interpretation of these findings as they may impact on legal issues. These include medical examiner investigations, driving under the influence and other transportation accident investigations, workplace pre-employment, random and for-cause drug testing and judicial monitoring of arrestees and parolees.

• For the most part, forensic toxicologists use commercial immunoassays directed primarily towards abused drugs. Commercial immunoassays developed for therapeutic monitoring of other drugs, veterinary drugs and pesticides, as well as immunoassays developed in research laboratories for specialized studies, may find a role in the forensic toxicology laboratory for specialized cases.

Immunoassays and Forensic Science Cont’d

• While most commercial immunoassays have been developed for a urine matrix, they have been applied by forensic toxicologists to other matrices, including blood, hair, saliva, sweat, tissue homogenates, blood stains and most other physiological samples that may be of value in the investigation. The nonurine matrix usually is much more complex in its composition. Sample pretreatments that range from simple deproteinations to multistep extractions to remove matrix components and/or concentrate the sample are often required. The heterogenous RIAs and ELISAs usually require less rigorous, if any, pretreatments (Abbot Diag.).

References

• Bell, Suzanne (2006). Forensic Chemistry. Upper Saddle River, New Jersey: Pearson Prentice Hall.

• Saferstein, Richard (2007). Criminalistics. Upper Saddle River, New Jersey: Pearson Prentice Hall.

• Wild, David (Ed.). (2005). The Immunoassay Handbook. Kidlington, Oxford: Elsevier.

• Evans, Susan (2004, June 15). Retrieved January 19, 2008, from SACB Online Web site: http://sacb.org.sg/

• Lee, Tim (2007, May 27). Antigens. Retrieved January 19, 2008, from Immunology Bookcase Web site: http://pim.medicine.dal.ca/atg.htm

• Monoclonal Antibody Production. Retrieved January 19, 2008, from Lecture Notes Web site: http://www.college.ucla.edu/webproject/micro7/lecturenotes/finished/monoclonal.html

• Masseyeff , RF (1991).Standardization of immunoassays.. Ann Ist Super Sanita. 27, 427-436.

• Moody, David E. (2006).Immunoassay in Forensic Toxicology. Encyclopedia of Analytical Chemistry .

• http://www.troopers.state.ny.us/Forensic_Science/Lab_Sections/Toxicology/

• http://www.abbottdiagnostics.com